Dynamic Disulfide Bond Topologies in von-Willebrand-Factor’s C4-Domain Undermine Platelet Binding

Abstract

Background: The von Willebrand Factor (vWF) is a key player in regulating hemostasis through adhesion of platelets to sites of vascular injury. It is a large multi-domain mechanosensitive protein stabilized by a net of disulfide bridges. Binding to platelet integrin is achieved by the vWF-C4 domain which exhibits a fixed fold, even under conditions of severe mechanical stress, but only if critical internal disulfide bonds are closed. Objective: To quantitatively determine C4's disulfide topologies and their implication in vWF's platelet-binding function via integrin. Methods: We employed a combination of classical Molecular Dynamics and quantum mechanical simulations, mass spectrometry, site-directed mutagenesis, and platelet binding assays. Results: We quantitatively show that two disulfide bonds in the vWF-C4 domain, namely the two major force-bearing ones, are partially reduced in human blood. Reduction leads to pronounced conformational changes within C4 that considerably affect the accessibility of the RGD-integrin binding motif, and thereby impair integrin-mediated platelet binding. Our combined approach also reveals that reduced species in the C4 domain undergo specific thiol/disulfide exchanges with the remaining disulfide bridges, in a process in which mechanical force may increase the proximity of specific reactant cysteines, further trapping C4 in a state of low integrin-binding propensity. We identify a multitude of redox states in all six vWF-C domains, suggesting disulfide bond reduction and swapping to be a general theme. Conclusion: Overall, our data put forward a mechanism in which disulfide bonds dynamically swap cysteine partners and control the interaction of vWF with integrin and potentially other partners, thereby critically influencing its hemostatic function.